Pharmaceutical compositions containing, as active ingredient, granulocyte-colony stimulating factor mutant protein or transferrin fusion protein thereof

ABSTRACT

The present invention relates to a fusion protein in which transferrin is peptide-bonded to a terminal of a granulocyte-colony stimulating factor (G-CSF) protein or a G-CSF mutant protein in which the 116 th  threonine is substituted with cysteine in the amino acid sequence of the G-CSF. Specifically, the granulocyte-colony stimulating factor (G-CSF) mutant protein of the present invention or the transferrin fusion protein thereof displays a significantly increased specific activity and blood stability, compared with the conventional human G-CSF, and has a higher purification efficiency than the conventional PEGylated G-CSF characterized by the extended half-life, so that it can be advantageously used for preventing or treating ischemic diseases or neutropenia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to PCT Application No.PCT/KR2014/011269 filed Nov. 21, 2014, entitled “PharmaceuticalComposition Containing, As Active Ingredient, GranulocytecolonyStimulating Factor Mutant Protein or Transferrin Fusion ProteinThereof,” which claims the benefit of and priority to Korean PatentApplication No. 10-2014-0093260, filed on Jul. 23, 2014 and KoreanPatent Application No. 10-2014-0137414, filed on Oct. 13, 2014. All theaforementioned applications are incorporated by reference herein intheir entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a fusion protein in which transferrinis peptide-bonded to a terminal of a granulocyte-colony stimulatingfactor (G-CSF) protein or a G-CSF mutant protein.

2. Description of the Related Art

Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein thatstimulates survival, proliferation, differentiation, and functions ofneutrophil and granulocyte progenitor cells and mature neutrophils.

The natural G-CSF which is now being used clinically is called‘Filgrastim’, and is a recombinant protein composed of 175 amino acidsoriginated from human amino acid sequence. This recombinant protein isexpressed in E. coli and is not glycosylated unlike the natural type.

G-CSF is used as an anticancer adjuvant for the prevention of infectiouscomplications caused by neutropenia accompanied by cancer treatment bystimulating neutrophil granulopoiesis. G-CSF demonstrates a beneficialclinical effect on cancer patients because it can reduce side effects offebrile neutropenia caused by chemo-therapy or radio-therapy for cancertreatment and thereby can reduce death rate by chemo-therapy for cancertreatment. G-CSF increases the number of hematopoietic progenitor cellsand accordingly reduces the side effects above.

It is also known that G-CSF stimulates bone marrow stem cells to moveischemic heart and accelerates differentiation of the stem cells intovascular cells and cardiomyocytes by stimulating myocardialregeneration.

The recombinant human G-CSF (rhG-CSF) displays pharmacological effectsonly for a short time. Therefore, it has to be administered at leastonce a day to treat ischemic disease or to treat leukopenia caused byanticancer chemo-therapy or radio-therapy. If a substance having a longin vivo half-life is administered, the administration times necessaryfor relieving leukopenia would be reduced and as a result it could bringthe effect of preventing infectious complications.

When Polyethylene glycol (PEG), the chemical polymer that is notdegraded in vivo, is fused to the N-terminal of G-CSF, a substancecalled ‘Pegfilgrastim’ is produced. This substance is clinically usedfor the treatment of leukopenia. This substance has an increased in vivohalf-life and displays a clinical effect while leukopenia continues evenwith the administration performed once or twice a week. However, in thatcase, a protein is fused with a chemical polymer by chemical reaction,and thus the problems of unsatisfactory fusion efficiency andcomplicated purification method can be caused.

Cysteine, the 17^(th) amino acid of G-CSF is exposed on the proteinsurface as nonsulfated binding state. In neutral pH, the exposedcysteine is combined with adjacent G-CSF cysteine via sulfide bond andas a result it loses its activity. A G-CSF mutant wherein the 17^(th)cysteine is substituted with serine displays increased stability inneutral pH, according to the previous reports.

Transferrin is the third most abundant protein in blood plasma, whichserves to transport iron ions present in the blood to various tissues.Transferrin has a relatively long half-life of 8 days, which is shorterthan that of albumin or immunoglobulin G. It enters the cell through thetransferrin receptor on the surface of the cell and once it suppliesiron ions, it is released to the outside of the cell in a state ofbinding with the receptor. Using these characteristics, transferrin hasbeen used as a fusion partner to increase circulating half-life bycombining the proteins with short half-lives of the prior art.

In this invention, threonine, the 116^(th) amino acid of human G-CSF,was substituted with cysteine to induce sulfide bonding with the 17^(th)cysteine of the original amino acid sequence of G-CSF. Sulfide bondingmakes the protein structure more stable so that the protein can beresistant against proteases, that is the protein now has a proteaseresistance. The attempt to have a protease resistance through theconstruction of such a mutant protein is new and the effect thereof hasnot been reported yet.

There has been no reports of using the mutant protein of G-CSF whereinthe 116^(th) amino acid is replaced with cysteine for the fusion withtransferrin to increase blood half-life of G-CSF.

The present inventors succeeded in making a fusion protein oftransferrin and the mutant protein of human G-CSF wherein the 116^(th)amino acid was replaced with cysteine and further confirmed that thefusion protein had significantly increased specific activity and bloodstability, compared with the unfused original human G-CSF, leading tothe completion of this invention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a fusion protein inwhich transferrin is peptide-bonded to a terminal of agranulocyte-colony stimulating factor (G-CSF) protein or a G-CSF mutantprotein in which the 116^(th) threonine is substituted with cysteine inthe amino acid sequence of the G-CSF.

To achieve the above object, the present invention provides a fusionprotein in which transferrin is peptide-bonded to a terminal of agranulocyte-colony stimulating factor (G-CSF) protein or a G-CSF mutantprotein in which the 116^(th) threonine is substituted with cysteine inthe amino acid sequence of the G-CSF, a G-CSF mutant protein in whichthe 116^(th) threonine is replaced with cysteine, and a use of the same.

The present invention also provides a pharmaceutical composition and ananticancer adjuvant for preventing or treating ischemic diseases orneutropenia comprising the fusion protein in which transferrin ispeptide-bonded to a terminal of a granulocyte-colony stimulating factor(G-CSF) protein or a G-CSF mutant protein in which the 116^(th)threonine is substituted with cysteine in the amino acid sequence of theG-CSF or the G-CSF mutant protein in which the 116^(th) threonine isreplaced with cysteine as an active ingredient.

The present invention also provides an expression vector containing thegene encoding a fusion protein in which transferrin is peptide-bonded toa terminal of a granulocyte-colony stimulating factor (G-CSF) protein ora G-CSF mutant protein in which the 116^(th) threonine is substitutedwith cysteine in the amino acid sequence of the G-CSF or a G-CSF mutantprotein in which the 116^(th) threonine is replaced with cysteine and atransformant prepared by inserting the expression vector above in a hostcell.

The present invention also provides a method for preparing theexpression vector containing the gene encoding a fusion protein in whichtransferrin is peptide-bonded to a terminal of a granulocyte-colonystimulating factor (G-CSF) protein or a G-CSF mutant protein in whichthe 116^(th) threonine is substituted with cysteine in the amino acidsequence of the G-CSF or a G-CSF mutant protein in which the 116^(th)threonine is replaced with cysteine, and the transformant prepared byinserting the expression vector above in a host cell.

The present invention also provides a method for preventing or treatingneutropenia or ischemic disease containing the step of administering thefusion protein in which transferrin is peptide-bonded to a terminal of agranulocyte-colony stimulating factor (G-CSF) protein or a G-CSF mutantprotein in which the 116^(th) threonine is substituted with cysteine inthe amino acid sequence of the G-CSF, the G-CSF mutant protein in whichthe 116^(th) threonine is replaced with cysteine, the expression vector,or the transformant above to a subject having neutropenia or ischemicdisease.

The present invention also provides a method for reducing neutrophilscontaining the step of administering the fusion protein in whichtransferrin is peptide-bonded to a terminal of a granulocyte-colonystimulating factor (G-CSF) protein or a G-CSF mutant protein in whichthe 116^(th) threonine is substituted with cysteine in the amino acidsequence of the G-CSF, the G-CSF mutant protein in which the 116^(th)threonine is replaced with cysteine, the expression vector, or thetransformant above to a subject.

In addition, the present invention provides a use of the fusion proteinin which transferrin is peptide-bonded to a terminal of agranulocyte-colony stimulating factor (G-CSF) protein or a G-CSF mutantprotein in which the 116^(th) threonine is substituted with cysteine inthe amino acid sequence of the G-CSF, the G-CSF mutant protein in whichthe 116^(th) threonine is replaced with cysteine, the expression vector,or the transformant above.

Advantageous Effect

The granulocyte-colony stimulating factor (G-CSF) mutant protein of thepresent invention or the transferrin fusion protein thereof displays asignificantly increased specific activity and blood stability, comparedwith the conventional human G-CSF, and has a higher purificationefficiency than the conventional PEGylated G-CSF characterized by theextended half-life, so that it can be advantageously used for preventingor treating ischemic diseases or neutropenia.

BRIEF DESCRIPTION OF THE DRAWINGS

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, wherein:

FIG. 1 is a diagram illustrating the cleavage map of pcDNA3.1(+)/preTfvector.

FIG. 2 is a diagram illustrating the cleavage map ofpcDNA3.1(+)/preTf(B) vector.

FIG. 3 is a diagram illustrating the cleavage map of pcDNA3.1(+)/Tf(B)vector.

FIG. 4 is a diagram illustrating the cleavage map ofpcDNA3.1(+)/G-CSF(T116C)-Tf vector.

FIG. 5 is a diagram illustrating the expression of G-CSF(T116C)-Tf inthe cell line Expi293F transfected with G-CSF(T116C)-Tf plasmid.

FIG. 6 is a diagram illustrating the G-CSF(T116C)-Tf protein separatedby DEAE Affi-gel blue chromatography.

FIG. 7 is a diagram illustrating the preparation, concentration, andseparation of Fe³⁺ fused G-CSF(T116C)-Tf in the form of holo(holoenzyme).

FIG. 8 is a diagram illustrating the binding force of G-CSF(T116C)-Tf inthe form of holo to the transferrin receptor.

FIG. 9 is a diagram illustrating the cell proliferation activity ofHL-60 cells treated with G-CSF, G-CSF-Tf, and G-CSF(T116C)-Tf.

FIG. 10 is a diagram illustrating the comparison of protease resistancebetween G-CSF and G-CSF(T116C).

FIG. 11 is a diagram illustrating the comparison of in vivo plasmahalf-life between G-CSF-Tf and G-CSF(T116C)-Tf.

FIG. 12 is a diagram illustrating the comparison of physiologicalactivity between G-CSF and G-CSF(T116C)-Tf in the rat havingneutropenia.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention is described in detail.

The present invention provides a fusion protein in which transferrin ispeptide-bonded to a terminal of a granulocyte-colony stimulating factor(G-CSF) protein or a G-CSF mutant protein in which the 116^(th)threonine is substituted with cysteine in the amino acid sequence of theG-CSF, a G-CSF mutant protein in which the 116^(th) threonine isreplaced with cysteine, and a use of the same.

The G-CSF or transferrin used in this invention can be originated fromanimals, plants, or microorganisms. It is more preferably humanoriginated G-CSF or transferrin herein, but it can be also aheterologous protein having the equal activity to human originated G-CSFor transferrin.

The protein above can additionally be modified such as phosphorylation,acetylation, methylation, glycosylation, etc., or it can be fused withanother protein. As long as the function of the protein is not lost,such mutant protein can be considered as the same as the protein beforeany modification.

Another amino acid that can replace the 116^(th) threonine is ahydrophobic amino acid, which is more precisely cysteine that ispossibly linked to the 17^(th) cysteine of G-CSF sequence via disulfidebond, but not always limited thereto.

The terminal where transferrin is bound can be either the amino terminal(5′-end, N-terminal) or the carboxy terminal (3′-end, C-terminal).

The said granulocyte-colony stimulating factor (G-CSF) protein is theprotein represented by SEQ. ID. NO: 1 but this sequence may haveaddition, deletion, or substitution of one or more amino acids in itssequence as long as the gene encoding the G-CSF is located at the sameposition on the chromosome and the activity is not changed.

The granulocyte-colony stimulating factor (G-CSF) protein abovepreferably has at least 80% homology, more preferably at least 90%homology, and most preferably at least 95%, 96%, 97%, 98%, 99%, or 99.5%homology to the amino acid sequence represented by SEQ. ID. NO: 1, butnot always limited thereto.

The said transferrin is the protein represented by SEQ. ID. NO: 2 butthis sequence may have addition, deletion, or substitution of one ormore amino acids in its sequence as long as the gene encoding thetransferrin is located at the same position on the chromosome and theactivity is not changed.

The transferrin protein above preferably has at least 80% homology, morepreferably at least 90% homology, and most preferably at least 95%, 96%,97%, 98%, 99%, or 99.5% homology to the amino acid sequence representedby SEQ. ID. NO: 2, but not always limited thereto.

The said granulocyte-colony stimulating factor (G-CSF) mutant protein isthe protein represented by SEQ. ID. NO: 3 but this sequence may haveaddition, deletion, or substitution of one or more amino acids in itssequence as long as the gene encoding the transferrin is located at thesame position on the chromosome and the activity is not changed.

The granulocyte-colony stimulating factor (G-CSF) mutant protein abovepreferably has at least 80% homology, more preferably at least 90%homology, and most preferably at least 95%, 96%, 97%, 98%, 99%, or 99.5%homology to the amino acid sequence represented by SEQ. ID. NO: 3, butnot always limited thereto.

The fusion protein in which transferrin is peptide-bonded to a terminalof G-CSF preferably has at least 80% homology, more preferably at least90% homology, and most preferably at least 95%, 96%, 97%, 98%, 99%, or99.5% homology to the amino acid sequence represented by SEQ. ID. NO: 4,but not always limited thereto.

The fusion protein in which transferrin is peptide-bonded to a terminalof a G-CSF mutant protein in which the 116^(th) threonine is substitutedwith cysteine in the amino acid sequence of the G-CSF preferably has atleast 80% homology, more preferably at least 90% homology, and mostpreferably at least 95%, 96%, 97%, 98%, 99%, or 99.5% homology to theamino acid sequence represented by SEQ. ID. NO: 5, but not alwayslimited thereto.

The said G-CSF, the G-CSF mutant protein in which the 116^(th) threonineis substituted with cysteine in the amino acid sequence of the G-CSF, orthe transferrin may have the substitution of nucleotide sequence forgene manipulation. For example the nucleotide sequence of a restrictionenzyme recognition site in the gene can be substituted with anothernucleotide sequence encoding another amino acid which is equal to theoriginal amino acid but not causing any changes in the protein activity,or a part of the gene terminal can be deleted, substituted or added witha restriction enzyme recognition site. For example, thymine of the BamHIrestriction enzyme recognition site of the protein gene, GGATCC, can besubstituted with cytosine, but not always limited thereto.

The said restriction enzyme is exemplified by EcoRI, BamHI, HindIII,kpnI, NotI, PstI, SmaI, XhoI, FokI, Alw26I, BbvI, BsrI, EarI, HphI,MboI, SfaNI, Tth111I, NaeI, NheI, NgoMIV, NheI, Eco57I, BcgI, BpI,Bsp24I, BaeI, CjeI, EcoPI, HintIII, and StyLTI, etc. However, anyrestriction enzyme that is used in the art can be used according to thegene, expression vector or genetic manipulation environment withoutlimitation.

The present invention also provides a pharmaceutical composition forpreventing or treating neutropenia and ischemic disease comprising thefusion protein in which transferrin is peptide-bonded to a terminal of aG-CSF mutant protein in which the 116^(th) threonine is substituted withcysteine in the amino acid sequence of the G-CSF or the G-CSF mutantprotein in which the 116^(th) threonine is substituted with cysteine inthe amino acid sequence of the G-CSF as an active ingredient, ananticancer adjuvant comprising the same, an expression vector comprisinga gene encoding the said protein, and a transformant prepared byinserting the said expression vector in a host cell.

The term ‘neutropenia’ in this invention indicates the abnormalreduction of neutrophils. When the number of blood neutrophils is lessthan 1500/μl, it is classified as light neutropenia and when the numberof blood neutrophils is less than 1000/μl, it is classified as moderateneutropenia. When the number of blood neutrophils is less than 500/μl,it is classified as severe neutropenia. In a large sense, leukopenia(neucopenia) is also included in neutropenia.

The ‘ischemic disease’ of the present invention is caused by cell damagecaused when the blood supply to the tissue is interrupted by hemorrhage,embolism, and infarction, etc, which is exemplified by trauma, graftrejection, stroke, cerebral infraction, ischemic renal disease, ischemiclung disease, infection mediated ischemic disease, ischemic limbdisease, ischemic cardiomyopathy, myocardial infarction, and heartfailure, etc.

The said another amino acid that can replace the 116^(th) threonine canbe a hydrophobic amino acid, which is precisely cysteine that issuitable for disulfide bond, but not always limited thereto.

The said granulocyte-colony stimulating factor (G-CSF) protein is theprotein represented by SEQ. ID. NO: 1 but this sequence may haveaddition, deletion, or substitution of one or more amino acids in itssequence as long as the gene encoding the G-CSF is located at the sameposition on the chromosome and the activity is not changed.

The granulocyte-colony stimulating factor (G-CSF) protein abovepreferably has at least 80% homology, more preferably at least 90%homology, and most preferably at least 95%, 96%, 97%, 98%, 99%, or 99.5%homology to the amino acid sequence represented by SEQ. ID. NO: 1, butnot always limited thereto.

The said transferrin is the protein represented by SEQ. ID. NO: 2 butthis sequence may have addition, deletion, or substitution of one ormore amino acids in its sequence as long as the gene encoding thetransferrin is located at the same position on the chromosome and theactivity is not changed.

The transferrin protein above preferably has at least 80% homology, morepreferably at least 90% homology, and most preferably at least 95%, 96%,97%, 98%, 99%, or 99.5% homology to the amino acid sequence representedby SEQ. ID. NO: 2, but not always limited thereto.

The said granulocyte-colony stimulating factor (G-CSF) mutant protein isthe protein represented by SEQ. ID. NO: 3 but this sequence may haveaddition, deletion, or substitution of one or more amino acids in itssequence as long as the gene encoding the transferrin is located at thesame position on the chromosome and the activity is not changed.

The granulocyte-colony stimulating factor (G-CSF) mutant protein abovepreferably has at least 80% homology, more preferably at least 90%homology, and most preferably at least 95%, 96%, 97%, 98%, 99%, or 99.5%homology to the amino acid sequence represented by SEQ. ID. NO: 3, butnot always limited thereto.

The fusion protein in which transferrin is peptide-bonded to a terminalof G-CSF preferably has at least 80% homology, more preferably at least90% homology, and most preferably at least 95%, 96%, 97%, 98%, 99%, or99.5% homology to the amino acid sequence represented by SEQ. ID. NO: 4,but not always limited thereto.

The fusion protein in which transferrin is peptide-bonded to a terminalof a G-CSF mutant protein in which the 116^(th) threonine is substitutedwith another amino acid in the amino acid sequence of the G-CSF but thissequence may have addition, deletion, or substitution of one or moreamino acids in its sequence as long as the gene encoding the G-CSF ortransferrin is located at the same position on the chromosome and theactivity is not changed.

The fusion protein in which transferrin is peptide-bonded to a terminalof a G-CSF mutant protein in which the 116^(th) threonine is substitutedwith another amino acid in the amino acid sequence of the G-CSFpreferably has at least 80% homology, more preferably at least 90%homology, and most preferably at least 95%, 96%, 97%, 98%, 99%, or 99.5%homology to the amino acid sequence represented by SEQ. ID. NO: 5, butnot always limited thereto.

The said G-CSF, the G-CSF mutant protein in which the 116^(th) threonineis substituted with another amino acid in the amino acid sequence of theG-CSF, or the transferrin may have the substitution of nucleotidesequence for gene manipulation. For example the nucleotide sequence of arestriction enzyme recognition site in the gene can be substituted withanother nucleotide sequence encoding another amino acid which is equalto the original amino acid but not causing any changes in the proteinactivity, or a part of the gene terminal can be deleted, substituted oradded with a restriction enzyme recognition site. For example, thymineof the BamHI restriction enzyme recognition site of the protein gene,GGATCC, can be substituted with cytosine, but not always limitedthereto.

The said restriction enzyme is exemplified by EcoRI, BamHI, kpnI, NotI,PstI, SmaI, XhoI, FokI, Alw26I, BbvI, BsrI, EarI, HphI, MboI, SfaNI,Tth111I, NaeI, NheI, NgoMIV, NheI, Eco57I, BcgI, BpI, Bsp24I, BaeI,CjeI, EcoPI, HintIII, and StyLTI, etc. However, any restriction enzymethat is used in the art can be used according to the gene, expressionvector or genetic manipulation environment without limitation.

The ‘expression vector’ of the invention is a tool to introduce thenucleic acid sequence encoding a target protein into a host cell, whichincludes plasmid, cosmid, BAC, and virus nucleic acid, etc. The saidvector generally includes a selection marker such as an antibioticresistant gene that can confirm the successful introduction of a targetgene into a host cell. The vector can further include promoter,operator, initiation codon, stop codon, polyadenylated sequence,enhancer, Kozak sequence, and Shine-Dalgarno sequence, etc, for theexpression of a target gene. If the vector is a replicable expressionvector, it can contain a replication origin.

The host cell herein can be selected from the group consisting of E.coli, yeast, fungi, plant cells, and animal cells, but not alwayslimited thereto and any host cell that is used for the production of arecombinant protein in this field can be accepted.

The pharmaceutical composition of the present invention can be preparedfor oral or parenteral administration by mixing with generally useddiluents or excipients such as fillers, extenders, binders, wettingagents, disintegrating agents and surfactant. Solid formulations fororal administration are tablets, pills, powders, granules and capsules.These solid formulations are prepared by mixing with one or moresuitable excipients such as starch, calcium carbonate, sucrose orlactose, gelatin, etc. Except for the simple excipients, lubricants, forexample magnesium stearate, talc, etc, can be used. Liquid formulationsfor oral administrations are suspensions, solutions, emulsions andsyrups, and the above-mentioned formulations can contain variousexcipients such as wetting agents, sweeteners, aromatics andpreservatives in addition to generally used simple diluents such aswater and liquid paraffin. Formulations for parenteral administrationare sterilized aqueous solutions, water-insoluble excipients,suspensions, emulsions, lyophilized preparations and suppositories.Water insoluble excipients and suspensions can contain, in addition tothe active compound or compounds, propylene glycol, polyethylene glycol,vegetable oil like olive oil, injectable ester like ethylolate, etc.Suppositories can contain, in addition to the active compound orcompounds, witepsol, macrogol, tween 61, cacao butter, laurin butter,glycerogelatin, etc.

The pharmaceutical composition of the present invention can beadministered orally or parenterally, and the parenteral administrationincludes skin external application, intrarectal injection, intravenousinjection, intramuscular injection, hypodermic injection, intrathoracicinjection, or intracerebroventricular injection.

The effective dosage of the pharmaceutical composition can be determinedaccording to weight, age, gender, health condition, diet, administrationfrequency, administration method, excretion and severity of disease, butthese cannot limit the present invention by any means. An individualdosage preferably contains the amount of active compound that issuitable for being administered in a single dose.

The single dose herein for the protein is 1˜100 mg, preferably 3˜50 mg,and more preferably 6˜30 mg, which can be administered once a day orseveral times a day and can be administered dividedly to differentparts.

The pharmaceutical composition of the present invention can beadministered alone or treated together with surgical operation, hormonetherapy, chemo-therapy and biological regulators.

The present invention also provides a method for preparing a fusionprotein in which transferrin is peptide-bonded to a terminal of agranulocyte-colony stimulating factor (G-CSF) protein or a G-CSF mutantprotein in which the 116^(th) threonine is substituted with cysteine inthe amino acid sequence of the G-CSF, a G-CSF mutant protein in whichthe 116^(th) threonine is replaced with cysteine, an expression vectorcontaining the gene encoding a fusion protein in which transferrin ispeptide-bonded to a terminal of a granulocyte-colony stimulating factor(G-CSF) protein or a G-CSF mutant protein in which the 116^(th)threonine is substituted with cysteine in the amino acid sequence of theG-CSF or a G-CSF mutant protein in which the 116^(th) threonine isreplaced with cysteine, and a transformant prepared by inserting theexpression vector above in a host cell.

The protein herein can be obtained by chemical synthesis or producedfrom natural cells, or purified from transfected host cells viarecombinant DNA technology. The obtained protein may have an additionalmodification such as phosphorylation, acetylation, methylation, andglycosylation, or can be as fused with another protein, but suchmodification is allowed as long as the function of the protein is notchanged.

The method for preparing the protein using recombinant DNA technology iscomposed of the following steps:

inserting a target gene for expression in an expression vector;

introducing the expression vector to a host cell; and

obtaining the protein produced by culturing the host cell.

However, the production of the protein is not limited to the above andany method known to those in the art can be used.

The step of obtaining the protein can be performed by using apurification technique well-known to those in the art, which isexemplified by protein precipitation, centrifugation, ultrasonicdisruption, ultrafiltration, dialysis, gel filtration, adsorptionchromatography, ion exchange chromatography, and affinitychromatography.

In a preferred embodiment of the present invention, a restriction enzymerecognition site was eliminated from the transferrin gene, and instead arestriction enzyme site was inserted in the 5′-end and also arestriction enzyme site and a stop codon were inserted in 3′-end,resulting in the construction of a plasmid. In the plasmid was insertedthe gene having the deletion of a restriction enzyme recognition sitefrom G-CSF sequence, resulting in the construction of a plasmid whereintransferrin gene was fused with G-CSF gene.

In a preferred embodiment of the present invention, a G-CSF mutant wasprepared by replacing the 116^(th) amino acid of G-CSF sequence withcysteine. A restriction enzyme recognition site was eliminated from thegene and instead a restriction enzyme site and Kozak sequence wereinserted in the 5′-end and a restriction enzyme site was also insertedin 3′-end, resulting in the construction of a plasmid. Then, transferringene was fused with the plasmid above to construct a plasmid whereintransferrin gene was fused with G-CSF mutant gene.

The present invention also provides a method for preventing or treatingneutropenia or ischemic disease containing the step of administering thefusion protein in which transferrin is peptide-bonded to a terminal of agranulocyte-colony stimulating factor (G-CSF) protein or a G-CSF mutantprotein in which the 116^(th) threonine is substituted with cysteine inthe amino acid sequence of the G-CSF, the G-CSF mutant protein in whichthe 116^(th) threonine is replaced with cysteine, the expression vectorharboring the gene encoding the said protein, or the transformantprepared by inserting the said expression vector to a host cell to asubject having neutropenia or ischemic disease.

The granulocyte-colony stimulating factor (G-CSF) mutant protein of thepresent invention or the transferrin fusion protein thereof displays asignificantly increased specific activity and blood stability, comparedwith the conventional human G-CSF, and has a higher purificationefficiency than the conventional PEGylated G-CSF characterized by theextended half-life, so that it can be advantageously used for preventingor treating ischemic diseases or neutropenia.

The present invention also provides a method for reducing neutrophilscontaining the step of administering the fusion protein in whichtransferrin is peptide-bonded to a terminal of a granulocyte-colonystimulating factor (G-CSF) protein or a G-CSF mutant protein in whichthe 116^(th) threonine is substituted with cysteine in the amino acidsequence of the G-CSF, the G-CSF mutant protein in which the 116^(th)threonine is replaced with cysteine, the expression vector, or thetransformant above to a subject.

The granulocyte-colony stimulating factor (G-CSF) mutant protein of thepresent invention or the transferrin fusion protein thereof displays asignificantly increased specific activity and blood stability, comparedwith the conventional human G-CSF, and has a higher purificationefficiency than the conventional PEGylated G-CSF characterized by theextended half-life, so that it can be advantageously used for reducingneutrophils

In addition, the present invention provides a use of the fusion proteinin which transferrin is peptide-bonded to a terminal of agranulocyte-colony stimulating factor (G-CSF) protein or a G-CSF mutantprotein in which the 116^(th) threonine is substituted with cysteine inthe amino acid sequence of the G-CSF, the G-CSF mutant protein in whichthe 116^(th) threonine is replaced with cysteine, the expression vector,or the transformant above.

The granulocyte-colony stimulating factor (G-CSF) mutant protein of thepresent invention or the transferrin fusion protein thereof displays asignificantly increased specific activity and blood stability, comparedwith the conventional human G-CSF, and has a higher purificationefficiency than the conventional PEGylated G-CSF characterized by theextended half-life, so that it can be advantageously used as ananticancer adjuvant, and used for preventing or treating ischemicdiseases or neutropenia.

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1: Plasmid Construction

<1-1> Construction of Transferrin Expression Plasmid

Total mRNA was obtained from the human hepatocellular carcinoma cellline HepG2. HepG2 cells were sub-cultured in DMEM (HyClone) supplementedwith 10% fetal bovine serum (WELGENE) and 1% penicillin-streptomycin(Gibco) in a 37° C., 5% CO₂ incubator. Two days later, the HepG2 cellculture medium was discarded and the cells were washed with phosphatebuffer (HyClone). The cell pellet was obtained, and the medium waseliminated by centrifugation. Then, the cell pellet was stored at −70°C. The frozen cells stored at −70° C. were thawed and total mRNA wasextracted therefrom by using RnaUs ToTal Tissue Premium RNA Preps(LeGene) with the standard conditions presented in the manual. cDNA wassynthesized from the obtained total mRNA by using oligo (dT) withPremium Express 1^(st) Strand cDNA synthesis system (LeGene).

PCR was performed by using the synthesized cDNA as a template with DNApolymerase (Phusion). The primers used in the reaction were custom-madein Cosmogenetech Co., Ltd. Particularly, NheI restriction enzyme siteand Kozak sequence were inserted in the 5′-end of the transferrin geneand Xho I restriction enzyme site and stop codon (TAA) were inserted inthe 3′-end of the gene. 10 pmol of a sense primer(5′-CATGCTAGCTCCACCATGAGGCTCGCCGTGGGAGCC-3′, SEQ. ID. NO: 9) and 10 pmolof an antisense primer (5′-AGACTCGAGTTAAGGTCTACGGAAAGTGCAG-3′, SEQ. ID.NO: 10) were used. The primers, DNA polymerase, and cDNA were mixed withbuffer, to which distilled water was added to make the total volume 50μl. PCR was performed as follows; predenaturation at 98° C. for 30seconds, denaturation at 98° C. for 10 seconds, annealing at 53° C. for30 seconds, polymerization at 72° C. for 1 minute, 30 cycles fromdenaturation to polymerization, and final extension at 72° C. for 10minutes. The amplified transferrin gene was transferred onto 1%agarose-gel for electrophoresis to confirm the size. The target area ofthe agarose-gel was cut off and DNA was purified by usingMEGAquick-spin™ Total Fragment DNA purification kit (iNtRoN).

The purified transferrin gene and pcDNA3.1(+) plasmid (Invitrogen) weredigested with the restriction enzymes NheI (Enzynomics) and XhoI(Enzynomics). 10 unit of the restriction enzyme and buffer 2 (10 mMTris-HCL pH 7.9, 50 mM NaCl, 10 mM MgCl₂, 1 mM DTT, 100 μg/ml BSA) wereadded thereto, followed by reaction at 37° C. for 2˜3 hours. Uponcompletion of the reaction, the size of the gene was confirmed by 1%agarose-gel electrophoresis. The target area of the agarose-gel was cutoff and DNA was purified by using MEGAquick-spin™ Total Fragment DNApurification kit (iNtRoN).

The purified transferrin gene and pcDNA3.1(+) vector were reacted withT4 DNA ligase (Takara) at 16° C. for 16 hours. Upon completion of thereaction, the vector was inserted in E. coli DH10B, which wasdistributed on Agar plate containing ampicillin, followed by culture at37° C. for 16 hours. Then, E. coli colony was selected.

To investigate whether or not the transferrin gene was successfullyinserted in pcDNA3.1(+) plasmid, PCR was performed by using DNApolymerase. The selected E. coli colony was diluted in water, which wasused as a template, followed by PCR with the sense primer(5′-CATGCTAGCTCCACCATGAGGCTCGCCGTGGGAGCC-3′, SEQ. ID. NO: 9) and theantisense primer (5′-AGACTCGAGTTAAGGTCTACGGAAAGTGCAG-3′, SEQ. ID. NO:10) used for the transferrin gene amplification above. PCR was performedas follows; predenaturation at 98° C. for 30 seconds, denaturation at98° C. for 10 seconds, annealing at 53° C. for 30 seconds,polymerization at 72° C. for 1 minute, 30 cycles from denaturation topolymerization, and final extension at 72° C. for 10 minutes. Uponcompletion of the PCR, electrophoresis on 1% agarose-gel was performedto confirm the gene.

The confirmed E. coli colony was inoculated in a LB liquid medium (1%trypton, 0.5% yeast extract, 1% NaCl), followed by culture at 37° C. for16 hours in a shaking incubator. The cultured E. coli was centrifuged toseparate the supernatant and the cell pellet. Plasmid DNA was purifiedfrom the cell pellet using Exprep™ Plasmid SV,mini (GeneAll). Sequencingof the purified plasmid was performed by Cosmogenetech Co., Ltd. Theprepared transferrin expression plasmid was named in this invention‘pcDNA3.1(+)/preTf (SEQ. ID. NO: 6)’ (FIG. 1).

To use the BamHI restriction enzyme site for the construction of aplasmid, the BamHI site on the transferrin gene was mutated bysite-direct mutagenesis to eliminate the BamHI restriction enzyme site.To eliminate the BamHI restriction enzyme site from the transferringene, thymine (T) was replaced with cytosine (C) but the amino acidsequence was maintained by aspartic acid. To replace the BamHIrestriction enzyme site, GGATCC, with the sequence GGACCC, the senseprimer (5′-CTATGGGTCAAAAGAGGACCCACAGACTTTCTATT-3′, SEQ. ID. NO: 11) andthe antisense primer (5′-AATAGAAAGTCTGTGGGTCCTCTTTTGACCCATAG-3′, SEQ.ID. NO: 12) custom-made in Cosmogenetech Co., Ltd. were used.Site-direct mutagenesis was performed using the preparedpcDNA3.1(+)/preTf plasmid as a template by using Muta-direct SiteDirected Mutagenesis kit (iNtRON) according to the manufacturer'sprotocol. Upon completion of the reaction, the gene was confirmed byelectrophoresis on 1% agarose-gel. The confirmed E. coli colony wasinoculated in a LB liquid medium containing ampicillin, followed byculture at 37° C. for 16 hours in a shaking incubator. The cultured E.coli was centrifuged to separate the supernatant and the cell pellet.Plasmid DNA was purified from the cell pellet using a plasmid extractionkit (GeneAll). The substitution of nucleic acid of the BamHI restrictionenzyme site on the transferrin gene was confirmed by sequencing. Theprepared plasmid was named in this invention ‘pcDNA3.1(+)/preTf(B) (SEQ.ID. NO: 7)’ (FIG. 2).

Transferrin is a secretary protein that contains a signal peptide tohelp the transportation of a protein synthesized on N-terminal to cellmembrane. Transferrin is composed of the amino acid sequence‘MRLAVGALLVCAVLGLCLA (SEQ. ID. NO: 13)’. To insert a humanleukocytopoiesis stimulating factor gene into the 5′-end of thetransferrin gene, the signal peptide of the transferrin was eliminatedand the restriction enzyme BamHI was placed in the 5′-end instead,resulting in the construction of a plasmid. In the 3′ end were insertedXhoI and Stop codon (TAA). PCR was performed by usingpcDNA3.1(+)/preTf(B) as a template in the presence of DNA polymerase.Xho I restriction enzyme site and stop codon (TAA) were inserted in the3′-end of the gene. PCR was performed by using pcDNA3.1(+)/preTf(B) as atemplate with DNA polymerase. The primers used in the reaction werecustom-made in Cosmogenetech Co., Ltd. 10 pmol of a sense primer(5′-CTCGGATCCGTCCCTGATAAAACTGTGAGATG-3′, SEQ. ID. NO: 14) and 10 pmol ofan antisense primer (5′-AGACTCGAGTTAAGGTCTACGGAAAGTGCAG-3′, SEQ. ID. NO:10) were used. The primers, DNA polymerase, and template were mixed withbuffer, to which distilled water was added to make the total volume 50μl. PCR was performed as follows; predenaturation at 98° C. for 30seconds, denaturation at 98° C. for 10 seconds, annealing at 53° C. for30 seconds, polymerization at 72° C. for 1 minute, 30 cycles fromdenaturation to polymerization, and final extension at 72° C. for 10minutes. The amplified transferrin gene was transferred onto 1%agarose-gel for electrophoresis to confirm the size. The target area ofthe agarose-gel was cut off and DNA was purified by usingMEGAquick-spin™ Total Fragment DNA purification kit (iNtRoN). Thepurified transferrin gene and pcDNA3.1(+) plasmid were digested with therestriction enzymes BamHI (Enzynomics) and XhoI (Enzynomics). 10 unit ofthe restriction enzyme and buffer 2 (10 mM Tris-HCL pH 7.9, 50 mM NaCl,10 mM MgCl₂, 1 mM DTT, 100 μg/ml BSA) were added thereto, followed byreaction at 37° C. for 2˜3 hours. Upon completion of the reaction, thesize of the gene was confirmed by 1% agarose-gel electrophoresis. Thetarget area of the agarose-gel was cut off and DNA was purified by usingMEGAquick-spin™ Total Fragment DNA purification kit (iNtRoN). Thepurified transferrin gene and pcDNA3.1(+) vector proceeded toelectrophoresis on 1% agarose-gel again, followed by DNA quantification.The transferrin gene and pcDNA3.1(+) vector were reacted with T4 DNAligase (Takara) at 16° C. for 16 hours. Upon completion of the reaction,the vector was inserted in E. coli DH10B, which was distributed on Agarplate containing ampicillin, followed by culture at 37° C. for 16 hours.Then, E. coli colony was selected. To investigate whether or not thetransferrin gene was successfully inserted in pcDNA3.1(+) plasmid, PCRwas performed. The selected E. coli colony was diluted in water, whichwas used as a template, followed by PCR with the sense primer(5′-CTCGGATCCGTCCCTGATAAAACTGTGAGATG-3′, SEQ. ID. NO: 14) and theantisense primer (5′-AGACTCGAGTTAAGGTCTACGGAAAGTGCAG-3′, SEQ. ID. NO:10) used for the transferrin gene amplification above. PCR was performedas follows; predenaturation at 98° C. for 30 seconds, denaturation at98° C. for 10 seconds, annealing at 53° C. for 30 seconds,polymerization at 72° C. for 1 minute, 30 cycles from denaturation topolymerization, and final extension at 72° C. for 10 minutes. Uponcompletion of the PCR, electrophoresis on 1% agarose-gel was performedto confirm the gene. The confirmed E. coli colony was inoculated in a LBliquid medium (1% trypton, 0.5% yeast extract, 1% NaCl), followed byculture at 37° C. for 16 hours in a shaking incubator. The cultured E.coli was centrifuged to separate the supernatant and the cell pellet.Plasmid DNA was purified from the cell pellet using Exprep™ PlasmidSV,mini (GeneAll). The insertion of the transferrin gene without signalpeptide was confirmed by sequencing. The prepared plasmid was named inthis invention ‘pcDNA3.1(+)/Tf(B) (SEQ. ID. NO: 8)’ (FIG. 3).

<1-2> Construction of G-CSF-Tf Expression Plasmid

To eliminate the BamHI restriction enzyme site from pcDNA6/G-CSF,pcDNA6/G-CSF plasmid was treated with 20 units of BamHI (Enzynomics),followed by reaction at 37° C. for 3 hours. The reacted plasmid (45 ng)was amplified by PCR with 10 pmol of a sense primer (5′-CATGCTAGCTCCACCATGGCTGGACCTGCCACCCAG-3′, SEQ. ID. NO: 15) and an antisense primer(5′-CATGGATCCGGGCTGGGCAAGGTGGCG-3′, SEQ. ID. NO: 16). As a result, aG-CSF gene fragment having Nhe I restriction enzyme site and Kozaksequence in the 5′-end and BamHI restriction enzyme site in the 3′-endwas prepared. 1 μg of the prepared G-CSF gene fragment and 365 ng ofpcDNA3.1(+)/Tf(B) plasmid were reacted with 10 units of NheI restrictionenzyme and 20 units of BamHI restriction enzyme respectively for at 37°C. for 4 hours. Electrophoresis was performed on DNA agarose-gel(0.5×TAE, 1% agarose). To construct pcDNA3.1(+)/G-CSF-Tf, the purifiedpcDNA3.1(+)/Tf(B) plasmid and the G-CSF gene fragment were mixed at themolar ratio of 1:3, which was reacted with 350 U of T4 DNA ligase at 25°C. for 1 hour. Then, E. coli DH5α was transfected with the reactionproduct. Colonies showing resistance against ampicillin were selected.Colony PCR was performed to confirm the construction ofpcDNA3.1(+)/G-CSF-Tf plasmid. Particularly, the colonies selected fromthe transfected E. coli DH5α were amplified by colony PCR with 10 pmolof the sense primer (5′-TAATACGACTCACTATAGGG-3′, SEQ. ID. NO: 17) andthe antisense primer (5′-AATAGAAAGTCTGTGGGTCCTCCTTTGACCCATAG-3′, SEQ.ID. NO: 18). The size of the amplified product was confirmed onagarose-gel (0.5×TAE, 1% agarose). The confirmed colony was inoculatedin a LB liquid medium (trypton 50 mg, yeast extract 25 mg, and sodiumchloride 50 mg per 5 ml) containing ampicillin (100 μg/ml), followed byculture for 15 hours. pcDNA3.1(+)/G-CSF-Tf was extracted by using aplasmid extraction kit. The successful construction ofpcDNA3.1(+)/G-CSF-Tf was confirmed by sequencing.

<1-3> Construction of G-CSF(T116C) and G-CSF(T116C)-Tf ExpressionPlasmid

10 pmol of the sense primer (5′-GCCGACTTTGCCACCTGCATCTGGCAGCAGAT-3′,SEQ. ID. NO: 19) and the antisense primer(5′-ATCTGCTGCCAGATGCAGGTGGCAAAGTCGGC-3′, SEQ. ID. NO: 20) were mixedwith pET21a(+)/G-CSF, followed by PCR. Site-directed mutagenesis wasperformed by the same manner as described in Example <1-1> to constructG-CSF(T116C). The resultant plasmid was named in this invention‘pET21a(+)/G-CSF(T116C)’.

The size of pET21a(+)/G-CSF(T116C) was confirmed by DNA agarose-gel(0.5×TAE, 1% agarose) electrophoresis. The target area of theagarose-gel was cut off and DNA was purified by using a gel purificationkit (Cosmogenetech). E. coli DH10B was transfected with the purifiedpET21a(+)/G-CSF(T116C). Colonies showing resistance against ampicillinwere selected. The selected colony was inoculated in a LB liquid medium(trypton 50 mg, yeast extract 25 mg, and sodium chloride 50 mg per 5 ml)containing ampicillin (100 μg/ml), followed by culture for 15 hours.pET21a(+)/G-CSF(T116C) was extracted by using a plasmid extraction kit.Sequencing was performed and as a result, the successful substitution ofthe nucleotide sequence was confirmed.

First, the sense primer (5′-GCCGACTTTGCCACCTGCATCTGGCAGCAGAT-3′, SEQ.ID. NO: 19) and the antisense primer(5′-ATCTGCTGCCAGATGCAGGTGGCAAAGTCGGC-3′, SEQ. ID. NO: 20) were dissolvedrespectively to the concentration of 10 pmol/μl. 10 pmol of each primerwas taken for PCR with 100 ng of the G-CSF gene (100 ng) inserted inpcDNA6. Site-directed mutagenesis was performed by the same manner asdescribed in Example <1-1> to obtain G-CSF(T116C) gene. The obtainedplasmid was named in this invention 'pcDNA6/G-CSF(T116C).

The size of pcDNA6/G-CSF(T116C) was confirmed by DNA agarose-gel(0.5×TAE, 1% agarose) electrophoresis. The target area of theagarose-gel was cut off and DNA was purified by using a gel purificationkit (Cosmogenetech). E. coli DH10B was transfected with the purifiedpcDNA6/G-CSF(T116C)). Colonies showing resistance against ampicillinwere selected. The selected colony was inoculated in a LB liquid medium(trypton 50 mg, yeast extract 25 mg, and sodium chloride 50 mg per 5 ml)containing ampicillin (100 μg/ml), followed by culture for 15 hours.pcDNA6/G-CSF(T116C) was extracted by using a plasmid extraction kit.Sequencing was performed and as a result, the successful substitution ofthe nucleotide sequence was confirmed. To eliminate the BamHIrestriction enzyme site from pcDNA6/G-CSF(T116C), 1 μg ofpcDNA6/G-CSF(T116C) plasmid was treated with 20 units of BamHI(Enzynomics), followed by reaction at 37° C. for 3 hours. The reactedplasmid (45 ng) was amplified by PCR with 10 pmol of a sense primer(5′-CATGCTAGCTCCACCA TGGCTGGACCTGCCACCCAG-3′, SEQ. ID. NO: 15) and anantisense primer (5′-CATGGATCCGGGCTGGGCAAGGTGGCG-3′, SEQ. ID. NO: 16).As a result, a G-CSF(T116C) DNA fragment having Nhe I restriction enzymesite and Kozak sequence in the 5′-end and BamHI restriction enzyme sitein the 3′-end was prepared. 1 μg of the prepared G-CSF(T116C) DNAfragment and 365 ng of pcDNA3.1(+)/Tf(B) plasmid were reacted with 10units of NheI restriction enzyme and 20 units of BamHI restrictionenzyme respectively for at 37° C. for 4 hours. Electrophoresis wasperformed on DNA agarose-gel (0.5×TAE, 1% agarose), followed bypurification by using a gel extraction kit. To constructpcDNA3.1(+)/G-CSF(T116C)-Tf, the purified pcDNA3.1(+)/Tf(B) plasmid andthe G-CSF(T116C) DNA fragment were mixed at the molar ratio of 1:3,which was reacted with 350 U of T4 DNA ligase at 25° C. for 1 hour.Then, E. coli DH5α was transfected with the reaction product. Coloniesshowing resistance against ampicillin were selected. Colony PCR wasperformed to confirm the construction of pcDNA3.1(+)/G-CSF(T116C)-Tfplasmid. Particularly, the colonies selected from the transfected E.coli DH5α were amplified by colony PCR with 10 pmol of the sense primer(5′-TAATACGACTCACTATAGGG-3′, SEQ. ID. NO: 17) and the antisense primer(5′-AATAGAAAGTCTGTGGGTCCTCCTTTGACCCATAG-3′, SEQ. ID. NO: 18). The sizeof the amplified product was confirmed on agarose-gel (0.5×TAE, 1%agarose). The confirmed colony was inoculated in a LB liquid medium(trypton 50 mg, yeast extract 25 mg, and sodium chloride 50 mg per 5 ml)containing ampicillin (100 μg/ml), followed by culture for 15 hours.pcDNA3.1(+)/G-CSF(T116C)-Tf was extracted by using a plasmid extractionkit. The successful construction of pcDNA3.1(+)/G-CSF(T116C)-Tf (SEQ.ID. NO: 21) was confirmed by sequencing.

Example 2: Protein Expression Through Transient Transfection

<2-1> Expression of G-CSF(T116C)

1 μl of pET21a(+)/G-CSF(T116C) plasmid was inoculated in 20 μl ofRosetta2 (DE3), the expression strain, followed by reaction at 42° C.for 90 seconds for transfection. The transfected expression strain wassmeared on LB plate containing ampicillin (50 μg/mL), followed byculture at 37° C. for 15 hours to obtain colonies. The colonies wereinoculated in 100 mL of LB medium containing ampicillin (50 μg/mL) andchloramphenicol (25 μg/mL), followed by culture at 37° C. for 15 hours.20 mL of the cultured strain was inoculated in 1 L of LB mediumcontaining ampicillin (50 μg/mL) and chloramphenicol (25 μg/mL),followed by culture with stirring at 37° C. for 4 hours until OD₆₀₀reached 0.8. 1 mM IPTG was added thereto, followed by stirring cultureat 37° C. for 4 hours. Centrifugation was performed at 6000 rpm for 15minutes to separate the supernatant and the precipitate respectively.The expression of the protein as an inclusion body was confirmed bySDS-PAGE.

<2-2> Expression of G-CSF(T116C)-Tf

The proteins used in this invention were expressed by extracellularsecretion using Expi293F™ (Gibco), the human embryonic kidney cancercell line modified for suspension culture. Expi293F™ cells included inExpi293F™ Expression system Kit (Gibco) were first thawed, which wereinoculated in a sterilized 125 mL flask (Thermo) containing 30 mL ofserum-free medium for animal cell culture (Gibco), followed by shakingculture at 125 rpm in a 37° C., 5% CO₂ incubator.

The density of Expi293F™ cell line was maintained at 3˜5×10⁶ cells/mL.Sub-culture was performed every 3˜4 days. After stabilized, the cellswere inoculated in serum-free medium at the density of 2×10⁶ cells/mLfor transfection, followed by shaking culture for 24 hours. Just beforethe transfection, the cell density was adjusted to 2×10⁶ cells/mL andthe cell survival rate was confirmed at least 90% before the followingexperiment.

22.5 μg of G-CSF-Tf and G-CSF(T116C)-Tf plasmid DNA was prepared anddiluted in Opti-MEM (Gibco). 45 μL of ExpiFectamine™ 293 Reagent 45 wasalso diluted in Opti-MEM®, which stood at room temperature for 5minutes. The plasmid and the ExpiFectamine™ 293 Reagent mixture werewell mixed, which stood at room temperature for 20 minutes. Then, themixture was evenly distributed to the cells. 16˜18 hours after thetransfection, ExpiFectamine™ 293 Transfection Enhancers 1 and 2 (Gibco)were added thereto in order to increase the protein expression, followedby additional culture for 32 hours under the same shaking culturecondition above. The obtained cell culture fluid was centrifuged at2,000×g for 10 minutes and as a result the supernatant was obtained.

To confirm the protein expression, the supernatant was mixed with 5×loading dye (Biosesang), which was boiled at 95° C. for 20 minutes. Themixture was loaded on 10% SDS-PAGE, followed by electrophoresis at 160 Vfor 1 hour. Upon completion of the electrophoresis, the protein on thegel was transferred onto PVDF Immobilon-P-Transfer Membrane (Millipore).The membrane was reacted in 5% skim milk at room temperature for 1 hour.After washing the membrane with washing buffer (phosphate buffercontaining 0.05% Tween 20), a transferrin antibody (2,000×, Santa Cruz)was added thereto, followed by reaction at room temperature for 2 hours.After washing the membrane with washing buffer again, a horseradishperoxides-conjugated antibody (10,000×, Santa Cruz) was added thereto,followed by reaction at room temperature for 1 hour. Upon completion ofthe antigen-antibody reaction, the membrane was washed with washingbuffer and color development was induced by using SuperSignal® West PicoChemiluminescent Substrate (Thermo). The molecular weight of G-CSF-Tfand G-CSF(T116C)-Tf was 105.7 kDa, suggesting that the protein wasnormally expressed (FIG. 5).

Example 3: Protein Purification

<3-1> Purification of G-CSF(T116C)

To purify the inclusion body, the cells were dissolved in a lysis buffer(50 mM Tris-Cl (pH 8.0), 2 mM EDTA, 1 mM PMSF), followed by cell lysisusing an ultrasonicator (lysis time: 5 minutes, ultrasonication: 30seconds, stand-by: 40 seconds). Centrifugation was performed at 10.000rpm for 30 minutes at 4° C. to eliminate the supernatant. Then, theprecipitate was dissolved in a washing buffer (50 mM Tris-Cl, pH 8.0, 2mM EDTA, 0.3% Triton) and lysed using an ultrasonicator (lysis time: 5minutes, ultrasonication: 30 seconds, stand-by: 40 seconds).Centrifugation was performed again at 10.000 rpm for 30 minutes at 4° C.to eliminate the supernatant. The precipitate was dissolved in a washingbuffer (50 mM Tris-Cl, pH8.0, 2 mM EDTA, 1 M NaCl) and lysed using anultrasonicator (lysis time: 5 minutes, ultrasonication: 30 seconds,stand-by: 40 seconds). Centrifugation was performed again at 10.000 rpmfor 30 minutes at 4° C. to eliminate the supernatant. The washedinclusion body was dissolved in a solubilization buffer (8 M Urea, 50 mMTris-Cl, pH8.0). 10 mL of the dissolved protein sample was loaded in adialysis tube, which was left in 2 L of a dialysis buffer (50 mM Tris-ClpH 8.0, 0.1% Tween20) at 4° C. for 16 hours for refolding. 2 M aceticacid was loaded to the reaction sample and pH was adjusted to 4.5,followed by centrifugation at 10.000 rpm for minutes at 4° C. Desaltingcolumn (17-5087-05) connected to Hiprep 26/10 AKTA prime plus FPLC wasfilled with a desalting buffer (25 mM Na-Acetate pH 4.5, 5% Sucrose,0.004% Tween20). Then, buffer exchange was performed by passing thesupernatant separated by centrifugation through the column above.

<3-2> Quantification of G-CSF(T116C)

The purified G-CSF(T116C) was quantified by using silver staining. Thestandard quantification curve was obtained by using grasin (less than 30ng/SDS-PAGE well), the G-CSF standard material. The unknown amount ofeach sample was compared to the standard quantification curve so as toquantify the protein.

<3-3> Quantification of G-CSF(T116C)-Tf

30 mL of the supernatant containing G-CSF-Tf or G-CSF(T116C)-Tf wasdialyzed in 4 L of 20 mM potassium phosphate buffer (pH 7.5), followedby buffer exchange. DEAE Affi-gel Blue column was filled with theG-CSF-Tf or G-CSF(T116C)-Tf included in the potassium phosphate bufferat the flow rate of 0.5 mL/min. Then, 72 mM of potassium phosphate wasspilled thereon via step gradient manner and thus the proteins absorbedon the resin were separated from other proteins included in the culturefluid (FIG. 6).

<3-4> Preparation of Iron (Fe³⁺) Fused Form

The preparation of the holo form of G-CSF(T116C)-Tf was based on [Zhanget al. BMC Biotechnology 2012, 12:92]. The purified G-CSF-Tf orG-CSF(T116C)-Tf was treated with the equivalent ferric ammonium citrate,followed by reaction at 37° C. for 2 hours. The mixture was dialyzed in2 L of phosphate buffered saline at 4° C. for 15 hours, during whichbuffer was exchanged. The sample was 10-fold concentrated by using aconcentrator (Millipore) (FIG. 7).

<3-5> Protein Quantification

The purified protein was quantified by Western blotting using atransferrin antibody. The measured transferrin was loaded in 10%SDS-PAGE at the concentrations of 15 ng, 20 ng, 25 ng, and 30 ng pereach well. Another purified protein whose concentration was notdetermined was loaded in another well, followed by electrophoresis.Western blotting was performed to quantify the protein samples bycomparing the purified protein with the transferrin standard curve.

Example 4: Investigation of Binding Force with Transferrin Receptor

Once transferrin is bound to the transferrin receptor expressed on thecell surface, it enters the cell via endocytosis. After releasing theiron, the transferrin-receptor complex moves to the cell surface againand thereafter it is secreted in blood. Transferrin repeats the processabove, that is the in vivo recycling metabolism, by which it suppliesthe iron and avoids being decomposed in the intracellular lysosome,resulting in the increased half-life. If a target protein having a shorthalf-life is conjugated with transferrin, the protein experiences the invivo recycling metabolism action of transferrin together withtransferrin. As a result, the target protein also can avoid beingdecomposed in vivo. So, the fusion protein in which G-CSF is conjugatedwith transferrin needs to maintain the binding force to transferrinreceptor in order to avoid in vivo degradation.

To investigate the binding force of the proteins of the invention to theextracellular surface transferrin receptor (TfR), the humanhepatocellular carcinoma cell line HepG2 demonstrating high expressionof TfR on the cell surface was used for the following experiment. HepG2cells were inoculated in a 96-well plate at the density of 5×10⁵cells/well, followed by culture for 24 hours. The HepG2 cells attachedon the plate were washed with phosphate buffer, to which serum-free DMEM(HyClone) supplemented with 1 mg/ml of BSA (Bovine Serum Albumin) wasadded. The mixture stood in a 37° C., 5% CO₂ incubator for 30 minutes toeliminate endogenous transferrin from the cells. The cells were washedwith phosphate buffer again, and then the test sample andfluorescence-labeled human transferrin Alexa Fluor® 647 (LifeTechnologies) were added to the serum-free DMEM supplemented with 1mg/ml of BSA at the concentrations of 133 nM and 5 μg/mL respectively.The mixture above was treated to the HepG2 cells prepared above, whichstood in a 37° C., 5% CO₂ incubator for 30 minutes. The protein sampleremaining in the medium was washed with cold phosphate buffer. Thetransferrin receptor conjugated fluorescence-transferrin was quantifiedby fluorimetry. For the fluorimetry, light source was given at 650 nmand fluorescence intensity at 668 nm was measured.

The transferrin receptor binding force of the holo form ofG-CSF(T116C)-Tf prepared in Example <3-4> was shown in FIG. 8. As aresult, as shown in FIG. 8, the transferrin receptor binding force ofthe G-CSF-Tf or G-CSF(T116C)-Tf fusion protein was similar to that ofthe natural human holo-transferrin (SIGMA). This result suggests thatthe G-CSF-Tf or G-CSF(T116C)-Tf fusion protein can have increasedhalf-life through experiencing the in vivo recycling due to theconjugation with the transferrin receptor, like human transferrin.

Example 5: Biological Activity of G-CSF Mutant Protein

To measure the biological activity of the purified G-CSF-Tf mutantprotein, the proliferation of HL-60 cells differentiated by 1.25% DMSOwas measured.

<5-1> Preparation and Treatment of Human Bone Marrow Derived Cell LineHL-60

The human bone marrow derived cell line HL-60 used in this invention wasdistributed from Korean Collection for Type Culture (KCTC), KoreaResearch Institute of Bioscience and Biotechnology (KRIBB). The cellline was cultured in RPMI-1640 containing 10% FBS in a 37° C., 5% CO₂incubator. The medium was replaced with a fresh one every 2˜3 days. Thecells were not anchorage-dependent. The reagents for the cell culturewere purchased from HyClone.

<5-2> Cell Proliferation Activity of G-CSF Mutant Protein

The number of cells was counted and the density was adjusted to be 2×10³cells/mL. The cells were treated with the differentiation inducer DMSO(dimethylsulfoxide, culture grade, SIGMA) at the final concentration of1.25% (v/v), followed by culture for days to induce the differentiationinto granulocytes. Before the test sample was added, the cells werecollected by centrifugation and washed with phosphate buffer (D-PBS).The cells were distributed in a 96-well plate (Corning), 100 μL per eachwell, at the density of 4×10⁴ cells/well.

The controls PEGylated human G-CSF (GenScript) and natural G-CSF(GenScript), and the test samples G-CSF-Tf, G-CSF(T116C)-Tf, andG-CSF(T116C) were diluted in desalting buffer (5 mM Sodium Phosphate, 5%Sucrose, 0.004% Tween-20), which were added to each well containing thedifferentiated HL-60 cells, followed by culture in a 37° C., 5% CO₂incubator for 72 hours. The final concentrations of each sample were300, 200, 100, 30, 15, 3, 0.3, and 0.03 ng/mL.

To investigate the cell proliferation level by the administration of thetest samples, 15 μL of CellTiter96™ (Promega) solution was added to eachwell, followed by culture for 4 hours. 100 μL of solubilization/stopsolution was added to each well to terminate the reaction. OD₅₇₀ of eachwell was measured with ELISA reader (TECAN). The increased number ofcells was counted and analyzed. The cell proliferation activity of thosetest samples was presented as the concentration equivalent to that ofG-CSF.

The cell proliferation activity of G-CSF, PEG.G-CSF, G-CSF(T116C),G-CSF-Tf, and G-CSF(T116C)-Tf is shown in FIG. 9. As shown in FIG. 9,the cell proliferation activity of G-CSF(T116C) was lower than that ofthe natural G-CSF but was similar to that of the PEGylated G-CSF. In themeantime, the cell proliferation activity of the G-CSF(T116C)-Tf fusionprotein in which transferrin is fused with a G-CSF(T116C) mutant proteinwas significantly increased, compared with that of G-CSF(T116C) and wasalso higher than that of the PEGylated G-CSF. Therefore, since theG-CSF(T116C)-Tf displays the higher cell proliferation activity thanthat of the commercial PEGylated G-CSF, it is expected to be a promisingcandidate as an agent for the treatment particularly using G-CSF.

Example 6: Resistance Against Blood Proteases

To measure the resistance against blood proteases of the G-CSF(T116C)mutant protein of the present invention, the protein was reacted withhuman serum and then the amount of G-CSF remaining in the serum for adesignated time was measured.

<6-1> Reaction of G-CSF with Human Serum

It was investigated whether or not the stability of the G-CSF mutantprotein of the present invention was associated closely with theresistance against blood protease. Particularly, human serum (SIGMA) wasinactivated at 56° C. for 30 minutes. 2.8 μg/ml of the protein samplewas added thereto at the ratio of 24:1 (v/v), followed by reaction at37° C. The reaction hours were 0, 3, 6, 9, 12, 15, and 18 hours.Complete protease inhibitor cocktail (Roche) was treated to the sampleat each designated hour, which was then stored at −70° C. uponcompletion of the reaction.

<6-2> Measurement of Residual G-CSF

After thawing the sample, the amount of G-CSF remaining after thereaction with human serum was quantified by sandwich ELISA (EnzymeLinked Immunosorbent Assay). Calibration curve of absorbance accordingto the protein concentration to the standard solution was prepared andregression analysis was performed to determine the G-CSF protein contentin the test solution.

The high protein binding microtiter plate (Corning) coated with a rabbitpolyclonal anti-G-CSF antibody (KOMA BIOTECH) at the concentration ofapproximately 2 μg/ml was prepared. 1× phosphate buffer containing 1%casein sodium salt was used as a blocking buffer. The standard solutionand the test solution were mixed with a buffer (10× diluted blockingbuffer). The protein sample and regents were distributed by 100 μl pereach well. The plate was treated with a washing buffer (1×PBS, 0.05%Tween-20) three times (300 μl/well) to wash out the non-reacted samples.The protein sample and the detection antibody (rabbit polyclonalanti-G-CSF antibody, KOMA BIOTECH) were treated thereto stepwise at roomtemperature for about 2 hours, followed by reaction withstreptavidin-horseradish peroxidase (Pierce) at 37° C. for 30 minutes.TMB (3,3′,5,5′-tetramethylbenzidine, Pierce) was treated thereto, whichstood at room temperature for about 7˜10 minutes and then colordevelopment was terminated with 2 M sulfuric acid. Then, OD₄₅₀ wasmeasured with a microplate reader (TECAN).

The absorbance was converted into concentration based on the calibrationcurve of protein concentration-absorbance to the standard solution. Thestandard curve was made using Excel program. The difference between themean value of the absorbance corresponding to each G-CSF standardsolution and the value of absorbance of the well treated with the bufferalone was calculated. For the determination of the test solutionconcentration, only when the absorbance value of each sample was in therange of the standard solution calibration curve, it was taken as valid.The absolute value (|R|) criterion calculated by using the correlationcoefficient (R²) as root (√) was set to at least 0.98. The amount of theremaining G-CSF protein (% G-CSF) was expressed by considering theconcentration of G-CSF (pg/ml) at reaction time point of 0 h as 100%.

The amount (%) of remaining G-CSF and G-CSF(T116C) by time period isshown in FIG. 10. The percentage of remaining G-CSF(T116C) wasapproximately 30% higher than that of natural G-CSF since 15 hours afterthe reaction started. It was confirmed that the disulfide bond betweenCys116 and Cys17 was helpful for the increase of resistance of theprotein against blood protease.

Example 7: Measurement of In Vivo Half-Life of G-CSF Mutant Proteins

To investigate the in vivo half-life of the G-CSF mutant protein and thetransferrin fusion protein of the invention, the proteins wereadministered to mice and then the level of G-CSF in the rat serum wasmeasured. The mentioned ‘in vivo half-life’ indicates the ‘half-life ofprotein in serum’. That is, the time point when the G-CSF protein levelwas down to 50% by the initial concentration during its circulation inserum was expressed numerically. Pharmacokinetic calculation wasperformed using PKsolver v2.0.

<7-1> Animal Test of G-CSF Mutant Protein

Animal test was performed to determine the duration of the CSF mutantprotein of the invention and its fusion protein in the body. All theprocedures such as purchase, breeding, administration and bloodcollection of test animals were performed by Qu-BEST Consulting Co.,Ltd.

For the test animals, 6-week-old ICR and SPF male mice were purchasedfrom Samtako. The mice were inspected and adapted for at least 6 days.On the last day of the adaptation period, individual weight wasmeasured. Based on the body weight, healthy animals without weight gainand general symptoms were selected and randomly arranged so that theaverage weight of each group was as equal as possible (35˜45 g peranimal). Each experimental group was assigned 6 animals.

On the day of the administration of the test sample, a unit dose foreach animal was calculated (5 mL/kg) based on the body weight measuredimmediately before the administration. The G-CSF mutant protein (20μg/kg) or the G-CSF mutant transferrin fusion protein (100 μg/kg) wasslowly injected into the Intravenous Bolus by using a disposable syringe(1 mL, 26G needle). The day of administration was defined as day 1 ofthe test.

After the injection, blood was drawn from the animals by orbitalcollection at the time point of 0.25, 0.5, 1, 3, 6, 12, 24, 48, and 72hours respectively. The blood sample was centrifuged at 10,000˜13,000rpm for 1˜2 minutes to separate plasma. The separated plasma was loadedin a tube (about 50 μL/tube) attached with a label containing theinformation of test type, animal number, and sampling time. The tube wasstored in a deep freezer (approximately −70° C.) until the day ofanalysis.

<7-2> Mouse Pharmacokinetics of G-CSF Mutant Proteins

The amount of active G-CSF in plasma was determined by ELISA afterthawing the sample on ice. The quantification of G-CSF in plasma byELISA was performed by using Human G-CSF ELISA Kit, pink-ONE (KomaBiotech.) according to the manual of the reagent. The calibration curveof the absorbance according to the protein concentration to the standardsolution was prepared and the G-CSF mutant content in the test solutionwas determined by regression analysis

Non-compartmental pharmacokinetic analysis was performed from the meanconcentration-time profile of each test compound by applying IV Bolusnon-compartment analysis input to PKsolver v2.0 program. Thepharmacokinetic parameters evaluate the terminal half-life (t_(1/2)).

The half-life of proteins in plasma is shown in FIG. 11. As shown inFIG. 11, G-CSF(T116C) mutant protein displayed a blood half-life similarto that of natural human G-CSF. This is a result of similar filtrationrate in the kidney, since the G-CSF(T116C) mutant protein has a similarprotein radius to the G-CSF. G-CSF-Tf protein showed an improved bloodhalf-life compared to G-CSF. This is because the fusion of transferrinto the G-CSF protein results in a significant increase in the proteinradius, resulting in a significantly lower renal filtration rate. TheG-CSF(T116C)-Tf fusion protein in which G-CSF(T116C) was fused totransferrin showed more extended half-life than that of G-CSF-Tf butdisplayed similar half-life to that of PEGylated G-CSF. Therefore, itwas suggested that when the G-CSF(T116C) mutant protein having theincreased resistance against blood protease was fused to transferrin,the renal filtration rate was decreased and the blood stability wasimproved, resulting in the maximization of in vivo blood half-life (FIG.11).

Example 8: Pharmacodynamics of G-CSF Mutant Proteins in Leukopenia Rats

To obtain the pharmacokinetic indices of the proteins of the presentinvention, hematologic analysis was performed using a rat disease model(Neutropenic Rat). All the procedures such as purchase, breeding,administration and hematologic analysis of test animals were performedby Qu-BEST Consulting Co., Ltd. The hematologic analysis items includeWBC, NEU, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, and Differential leucocytecount, etc.

5-week-old SPF Sprague Dawley male rats were adapted at least 5 days andthen randomly allocated into four groups. Each rat was in the weightrange of 350˜400 g. One day before the administration of the testsample, cyclophosphamide (90 mg/kg) was intraperitoneally administeredonce to induce leukopenia (Neutropenic). The SD rats were intravenouslyadministered with G-CSF once, followed by hematologic tests for 5 days.The test compound was diluted in PBS (Phosphate Buffered Saline) to makethe final concentration of 0.004 mg/mL. On the day of the administrationof the test sample, a unit dose for each animal was calculated (2 mL/kg)based on the body weight measured immediately before the administration.The test sample was slowly injected into the Intravenous Bolus by usinga disposable syringe (1 mL, 26G needle). The day of administration wasdefined as day 1 of the test.

Before the administration and 6, 12, 24, 48, and 72 hours after theadministration, blood collection (approximately 200 μl) was performed inthe jugular vein using a disposable syringe (1 mL, 26G needle). Theblood was stored in the tube treated with an anticoagulant (5% sodiumEDTA) to prevent the blood from coagulation. The components in the bloodwere analyzed using an automatic blood analyzer in a refrigerated statewithin 3 hours. The results are presented with mean value and standarddeviation. The difference between the control and each experimentalgroup was compared statistically by using GraphPad Prism by One-wayANOVA and Dunnett's test.

Time-dependent neutrophil levels after the administration of G-CSF-Tfand its mutant proteins were presented in FIG. 2. As shown in FIG. 12,from the comparison of the neutrophil levels among the test samples, itwas confirmed that the G-CSF(T116C)-Tf administered group displayedhigher absolute neutrophil count than the G-CSF and PEGylated G-CSFadministered groups after 6 and 12 hours from the administration. Theabsolute neutrophil count of G-CSF was a little higher than that of thecontrol 24 hours after the administration, which was though notsignificant. In the meantime, PEGylated G-CSF and G-cSF(T116C)-Tfdisplayed the increased WBC (white blood cell) and neutrophil levels inthe rats having leukopenia induced by cyclophosphamide up to 24 hoursfrom the administration. G-CSF(T116C)-Tf fusion protein was circulatedin vivo by using transferrin as a mediator, during which in vivoactivity was maintained longer than G-CSF and was as high as that ofPEGylated G-CSF. On the other hand, the pharmacokinetic parameters (Cmaxand AUC0-t) of the fusion protein were higher than those of G-CSF, asshown in FIG. 9. This seemed to be because the neutrophilic precursorcell proliferation was greater in the protein than in PEGylated G-CSF inthe same length of period (FIG. 12).

INDUSTRIAL APPLICABILITY

The induced material in this invention exhibits G-CSF like biologicalactivity that has been used clinically, so that the material of thepresent invention can be used for the treatment of ischemic disease oras an anticancer adjuvant for the prevention of infectious complicationscaused by neutropenia resulted from chemo-therapy or radio-therapy forthe treatment of cancer due to its activity of stimulating neutrophilgranulopoiesis.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended Claims.

What is claimed is:
 1. A fusion protein in which transferrin is peptide-bonded to a G-CSF (granulocyte-colony stimulating factor) mutant protein in which the 116th threonine is substituted with a cysteine.
 2. The fusion protein according to claim 1, wherein the transferrin has the deletion of a signal peptide at the amino terminal region.
 3. A method, comprising administering the fusion protein of claim 2 to a subject.
 4. The fusion protein according to claim 1, wherein the substituted cysteine forms a disulfide bond with the 17th cysteine of the G-CSF mutant protein.
 5. A method, comprising administering the fusion protein of claim 4 to a subject.
 6. A method, comprising administering the fusion protein of claim 1 to a subject.
 7. A fusion protein, comprising a transferrin protein that is peptide-bonded to a G-CSF (granulocyte-colony stimulating factor) mutant protein in which the 116th threonine is a substituted with a cysteine.
 8. The fusion protein according to claim 7, wherein the transferrin protein does not have a native signal peptide at the amino terminal region.
 9. A method, comprising administering the fusion protein of claim 7 to a subject.
 10. A fusion protein, comprising: a G-CSF (granulocyte-colony stimulating factor) mutant protein in which the 116th threonin is substituted with a cysteine; and a transferrin protein peptide-bonded to the G-CSF mutant protein, wherein the transferrin has deletion of a signal peptide at the amino terminal region.
 11. A method, comprising administering the fusion protein of claim 10 to a subject. 